Prolonged intraocular residence and retinal tissue distribution of a fourth-generation compstatin-based C3 inhibitor in non-human primates

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Abstract

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss among the elderly population. Genetic studies in susceptible individuals have linked this ocular disease to deregulated complement activity that culminates in increased C3 turnover, retinal inflammation and photoreceptor loss. Therapeutic targeting of C3 has therefore emerged as a promising strategy for broadly intercepting the detrimental proinflammatory consequences of complement activation in the retinal tissue. In this regard, a PEGylated second-generation derivative of the compstatin family of C3-targeted inhibitors is currently in late-stage clinical development as a treatment option for geographic atrophy, an advanced form of AMD which lacks approved therapy. While efficacy has been strongly suggested in phase 2 clinical trials, crucial aspects still remain to be defined with regard to the ocular bioavailability, tissue distribution and residence, and dosing frequency of such inhibitors in AMD patients. Here we report the intraocular distribution and pharmacokinetic profile of the fourth-generation compstatin analog, Cp40-KKK in cynomolgus monkeys following a single intravitreal injection. Using a sensitive surface plasmon resonance (SPR)-based competition assay and ELISA, we have quantified both the amount of inhibitor and the concentration of C3 retained in the vitreous of Cp40-KKK-injected animals. Cp40-KKK displays prolonged intraocular residence, being detected at C3-saturating levels for over 3 months after a single intravitreal injection. Moreover, we have probed the distribution of Cp40-KKK within the ocular tissue by means of immunohistochemistry and highly specific anti-Cp40-KKK antibodies. Both C3 and Cp40-KKK were detected in the retinal tissue of inhibitor-injected animals, with prominent co-localization in the choroid one-month post intravitreal injection. These results attest to the high retinal tissue penetrance and target-driven distribution of Cp40-KKK. Given its subnanomolar binding affinity and prolonged ocular residence, Cp40-KKK constitutes a promising drug candidate for ocular pathologies underpinned by deregulated C3 activation.

Introduction

AMD is a prevalent ocular disease with complement-driven pathophysiology that can lead to irreversible vision loss in affected elderly individuals [1]. It progresses from its early form, marked by accumulation of lipid and protein-rich deposits (drusen) within Bruch's membrane and resulting sub-retinal inflammation, to the advanced dry form (geographic atrophy, GA) in which there is loss of choroidal vessels, retinal pigment epithelium (RPE), and photoreceptors [1]. Dry AMD can evolve into a wet (neovascular) form characterized by aberrant neovascularization with vessel leakage and further inflammatory damage often leading to retinal atrophy and vision loss [[1], [2]]. Advanced dry AMD (GA) still lacks approved treatment, while the current standard of treatment for wet AMD is locally administered anti-vascular endothelial growth factor (VEGF) therapy [1].

Over a decade ago, the discovery that a common genetic variant (p.Y402H) of the complement regulator factor H (FH) predisposes individuals to AMD drastically reshaped our perception of how the complement system modulates disease progression in AMD and other retinal diseases driven by deregulated complement activation [3,4]. Extensive genetic studies have identified several risk-associated common or rare genetic variants in different components and regulatory proteins of the alternative complement pathway (AP) [1,5]. Whereas the precise role of complement deregulation in the pathophysiology of AMD still remains ill-defined, there is compelling evidence that a deregulated AP response leading to increased C3 turnover and retinal inflammation is integrally involved in the early stages of this disease [6]. This growing appreciation of complement's involvement in AMD pathology has ignited efforts to target this system therapeutically by tapping into various druggable targets of the cascade [6,7].

Given the disease-exacerbating role of the AP, initial efforts to develop therapeutics for treating GA focused on factor D (FD), the rate-limiting protease of this pathway, responsible for the formation of the AP C3 convertase. However, the failure of the FD-targeting mAb, lampalizumab (Genentech/Roche) to meet its primary endpoint in two multicenter phase III trials signified a setback in the clinical development of FD inhibitors for AMD [8]. At the same time these trials raised awareness about possible gaps in the biology of FD's involvement in AMD and phenomena that may curb clinical efficacy, such as, reduced drug bioavailability, insufficient tissue penetration or potential FD by-pass pathways that may become operative in a prolonged therapeutic regimen. Despite this clinical setback, the pharmaceutical industry has rekindled its efforts to develop ocular therapeutics focused on alternative targets, including factor B (FB) and C5 inhibitors [7,9]. Interestingly, Zimura/avacincaptad pegol, a PEGylated anti-C5 aptamer developed by Iveric (formerly Opthotech) has shown promising results in a phase IIb trial in GA patients with significant reduction in GA lesion size [10], thus expanding the toolbox of drug candidates for AMD.

While these approaches have shown early clinical promise, the therapeutic targeting of C3 activation has gained considerable traction in recent years as a more comprehensive strategy whereby all pathways are broadly inhibited at the level of C3, regardless of initiating triggers or downstream effector mechanisms [9]. C3 inhibition can singlehandedly afford therapeutic coverage against multiple pathogenic drivers in AMD by preventing generation of both C3 and C5-derived fragments that modulate phagocytic cell recruitment, oxidative tissue damage, inflammatory cell activation and cytolytic activity via membrane attack complex (MAC) assembly [11,12].

In this respect, peptidic C3 inhibitors of the compstatin family have entered clinical development as promising ocular therapeutics [6,7]. 4(1MeW)7W/POT-4, a second-generation compstatin analog was initially evaluated in a phase I study in wet AMD patients showing good safety and tolerability (Potentia/Alcon) [13]. Despite the lack of clinical efficacy in phase II trials in wet AMD, likely because of insufficient dosing [14], initial studies with POT-4 propelled the development of its PEGylated version, APL-2/pegcetacoplan (Apellis). This C3 therapeutic has recently completed a phase II trial in GA patients having shown safety and therapeutic efficacy in terms of reducing GA lesion size independently of genetic variants that can skew GA progression [15]. Pegcetacoplan is currently being evaluated in two multi-center phase III studies in GA patients in monthly or bimonthly dosing regimens [16]. While PEGylation has likely increased the intraocular retention of APL-2, as compared to POT-4, the presence of a fraction of patients with wet AMD conversions in the phase II trial raises the possibility that at high PEG burdens, PEG–triggered choroidal neovascularization (CNV) or other mechanisms could modulate the clinical outcome [17,18]. The incidence of a similar CNV conversion rate in GA patients receiving the PEGylated therapeutic Zimura provides further evidence for this scenario, suggesting that alternative PEG-free formulations should be explored for delivering complement inhibitors intraocularly [19] [10].

The development of third and fourth-generation non-PEGylated compstatins with improved target affinity, solubility and favourable pharmacokinetic (PK) profiles may confer benefits in terms of improved efficacy, reduced dosing frequency and lower risk for PEG-related adverse events in AMD patients [7,13,20]. AMY-101 (Amyndas), a C3 therapeutic based on the third-generation compstatin analog Cp40 [21], is currently evaluated in phase IIa trials in patients with periodontal disease, and is clinically developed for a spectrum of renal and hemolytic indications, further illustrating the clinical feasibility of this approach [[22], [23], [24], [25]]. Here we report the intraocular pharmacokinetic profile and retinal tissue distribution of the fourth-generation compstatin analog, Cp40-KKK [20]. Its prolonged intraocular residence at C3-saturating levels, for over 3 months after a single intravitreal (IVT) injection, makes it a suitable drug candidate for ocular indications associated with chronic C3 dysregulation.

Section snippets

Inhibitors and reagents

The compstatin analog Cp40 (dTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH2, 1.8 kDa) and two derivatives containing either two or three lysine residues at the C-terminus (Cp40-KK and Cp40-KKK) was synthesized by solid-phase peptide synthesis, cyclized by disulfide bridge formation, and purified as previously described [20,21]. Plasma-purified human C3 was purchased from Complement Techology (Tyler, TX). Pooled EDTA-human plasma was purchased from Innovative Research Inc.

Prolonged residence time of compstatin analogs Cp40-KK and Cp40-KKK in the vitreous of non-human primates

The advancement of the PEGylated C3-targeted therapeutic APL-2/pegcetacoplan (Apellis) to phase III trials in diseases of the hemolytic and ocular spectrum (i.e., PNH and GA/AMD) has marked an important milestone in the path towards clinical C3 inhibition [6,27,28]. Clinical results are now validating the safety and efficacy of this long-debated targeting strategy, thereby eliminating the purported risk of compromised pathogen immunosurveillance during chronic anti-C3 treatment [7]. While a

Declaration of Competing Interest

J.D. Lambris is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors for therapeutic purposes. J.D. Lambris and D. Ricklin are inventors of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. J.D. Lambris is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals (i.e., 4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives such as

Acknowledgements

We thank Prof. Ronald P. Taylor (University of Virginia) for generously providing the anti-C3b/iC3b monoclonal antibody, D131-45A-8E11, for intravitreal C3 measurements. This work was supported by grants from the U.S. National Institutes of Health (AI068730; to JDL). DCM acknowledges support from project MIS 5002559 which is implemented under the “Action for the Strategic Development on the Research and Technological Sector”, funded by the Operational Programme “Competitiveness,

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    These authors contributed equally to this study.

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